van der Geer P, Wiley S, Gish G D, Pawson T
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Curr Biol. 1996 Nov 1;6(11):1435-44. doi: 10.1016/s0960-9822(96)00748-8.
Signal transduction initiated by a wide variety of extracellular signals involves the activation of protein-tyrosine kinases. Phosphorylated tyrosine residues in activated receptors or docking proteins then function as binding sites for the Src homology 2 (SH2) or phosphotyrosine-binding (PTB) domains of cytoplasmic signalling proteins. Shc is an adaptor protein that contains both PTB and SH2 domains and becomes phosphorylated on tyrosine in response to many different extracellular stimuli. These results have suggested that Shc is a prominent effector of protein-tyrosine kinase signalling. Thus far, only a single Shc phosphorylation site, the tyrosine at position 317 (Y317) has been identified. Phosphorylation of Y317 has been implicated in Grb2 binding and activation of the Ras pathway.
Here, we report the identification of two major and novel Shc tyrosine phosphorylation sites, Y239 and Y240. These residues are present in the central proline-rich (CH1) region and are conserved in all isoforms of Shc. Y239/240 are co-ordinately phosphorylated by the Src protein-tyrosine kinase in vitro, and in response to epidermal growth factor stimulation or in v-src-transformed cells in vivo. Mutagenesis studies indicate that Y239/240 make an important contribution to the association of Shc with Grb2. Phosphopeptide-binding studies suggest that these two tyrosine residues may be involved in interactions with a number of cellular proteins.
Shc is the most prominent general substrate for protein-tyrosine kinases in vivo. The identification of two novel Shc phosphorylation sites indicates that Shc has the potential to interact with multiple downstream effectors. Shc Y239/240 are highly conserved in evolution, suggesting that the phosphorylation of these residues is of fundamental importance. We propose that distinct Shc phosphorylation isomers from different signalling complexes and thereby activate separate downstream signalling cascades.
由多种细胞外信号引发的信号转导涉及蛋白酪氨酸激酶的激活。活化受体或对接蛋白中磷酸化的酪氨酸残基随后作为细胞质信号蛋白的Src同源2（SH2）或磷酸酪氨酸结合（PTB）结构域的结合位点。Shc是一种衔接蛋白，包含PTB和SH2结构域，在受到许多不同的细胞外刺激时会在酪氨酸上发生磷酸化。这些结果表明Shc是蛋白酪氨酸激酶信号传导的重要效应器。到目前为止，仅鉴定出一个Shc磷酸化位点，即317位酪氨酸（Y317）。Y317的磷酸化与Grb2结合及Ras途径的激活有关。
在此，我们报告鉴定出两个主要的新型Shc酪氨酸磷酸化位点，Y239和Y240。这些残基存在于富含脯氨酸的中央（CH1）区域，并且在Shc的所有同工型中都保守。Y239/240在体外被Src蛋白酪氨酸激酶协同磷酸化，并在体内对表皮生长因子刺激或v-src转化细胞产生反应时被磷酸化。诱变研究表明，Y239/240对Shc与Grb2的结合起重要作用。磷酸肽结合研究表明，这两个酪氨酸残基可能参与与许多细胞蛋白的相互作用。
Shc是体内蛋白酪氨酸激酶最主要的一般底物。两个新型Shc磷酸化位点的鉴定表明，Shc有可能与多个下游效应器相互作用。Shc Y239/240在进化过程中高度保守，表明这些残基的磷酸化至关重要。我们提出，来自不同信号复合物的不同Shc磷酸化异构体从而激活不同的下游信号级联反应。
